The goal of this project is to further investigate the regulatory pathways that control virulence expression in Vibrio cholerae, a gram-negative bacterium that causes the human diarrheal disease called cholera. It also is a model organism for understanding bacterial pathogenesis, with the identification of many of its virulence factors and requirements for colonization. Yet, much remains unknown with regards to the organism's mechanisms to sense the environment and respond to virulence activating stimuli. Taking a chemical genetics approach to studying V. choIerae pathogenesis, the candidate has identified 11 novel small molecules that inhibit activation of the virulence genes in V. cholerae. Preliminary studies of these compounds suggest that they define regulatory mechanisms for virulence activation that have not been previously described, including mechanisms to respond to anaerobic conditions and to coordinate virulence expression with biofilm formation. The 11 compounds will be used to identify and characterize these novel pathways. In addition to genetic and biochemical approaches, two new methods will be developed for identifying regulators of virulence expression in V. cholerae. These two methods, a complementation method using a transposon based inducible promoter system and a bacterial-3-hybrid system for identifying small molecule-protein interactions, can be applied to the study of other bacterial systems. The long-term goal of the candidate is to merge the powerful fields of chemical and molecular genetics in the study of bacterial pathogenesis. In this era of increasing microbial resistance to current antibiotics, understanding the basic mechanisms of pathogenesis is critical to finding new solutions. Small molecule effectors and inhibitors may be developed as tools to probe the biology of microbes, providing insight into the organism's requirements for survival in the environment as well as for causing disease in the host. The short-term goal of the candidate is to acquire the skills of molecular bacterial genetics and postgenomic analysis in the laboratory of the sponsor, Dr. John Mekalanos, thus allowing her to become a productive, independent physician-scientist.